Potentiostatic preparation of molecular adsorbates for scanning probe microscopy

ABSTRACT

.[.A method of.]. .Iadd.An apparatus and method for .Iaddend.preparing molecular adsorbates for scanning probe microscopy by potentiostatic methods. Negatively charged molecules are deposited upon and held to a substrate with an electrochemical cell having a gold substrate, a platinum wire counter electrode and a silver wire reference electrode. The polymer to be observed is dissolved into a buffer solution which is non-reactive with the substrate which is gold (111).

INTRODUCTION

.[.This.]. .Iadd.The .Iaddend.present invention relates generally toscanning probe microscopy and more particularly to .Iadd.an apparatusand method for .Iaddend.the potentiostatic preparation of molecularadsorbates for study with scanning probe microscopes.

.Iadd.This invention was made with Government support under contract No.N00014-90-J-1655 awarded by the Department of the Navy and grant DIR89-20053 awarded by the National Science Foundation. The Government hascertain rights in the invention..Iaddend.

BACKGROUND OF THE INVENTION

Various ways have heretofore been proposed for chemically reactingmolecules with a metal substrate in an electrochemistry cell. In somecases, the prior methodology allows molecules to be bonded stronglyenough so that they can be imaged in a scanning tunnelling microscope(STM) or in .[.the.]. .Iadd.an .Iaddend.atomic force microscope (AFM).However, in the case of negatively charged molecules, such as DNA, it isextremely difficult to get them to adhere to an electrode because mostmetal surfaces are intrinsically negatively charged and, as such, repelthe molecule. Thus a clear need exists for new and improved technologyfor the potentiostatic preparation of negatively charged .[.molecules.]..Iadd.molecular .Iaddend.adsorbates such as DNA to enable them to beproperly bonded to .Iadd.a .Iaddend.suitable substrate so .Iadd.that.Iaddend.they can be imaged in .Iadd.a .Iaddend.scanning tunnellingmicroscope .[.(STM).]. or .Iadd.an .Iaddend.atomic force .[.microscopes(AFM).]. .Iadd.microscope.Iaddend.. It is .[.toward.]. .Iadd.to.Iaddend.this end that the present invention is directed.

BRIEF SUMMARY OF THE INVENTION

The present invention is predicated upon the discovery of a remarkablysimple procedure for getting negatively charged molecules onto asubstrate and holding them there. The new methodology is based in parton the concept that DNA (or any other negatively charged molecule) canbe attracted to a surface which is positively charged by virtue of itsinteraction with an electrolyte (.[.See: Lindsay et al, 1988.]..Iadd.See: Lindsay, S. M., and Barris, B., "Imaging DNA Molecules on aMetal Surface Under Water by STM", Journal of Vacuum Science andTechnology. Vol. A6, Pages 544-547 (1988)).Iaddend.. What is new andunexpected is that the same forces that attract the molecules to thesurface are capable, in the practice of the present invention, ofholding such molecules in place on that surface for study in an.[.SPM.]. .Iadd.STM .Iaddend.or an AFM. More particularly, the presentinvention relates to the potentiostatic preparation of molecularadsorbates for scanning probe microscopy in an electrochemical cellhaving a gold substrate, a platinum wire counter electrode and a silverwire reference electrode placed upon the microscope. The polymer to beobserved is dissolved into a buffer solution which is non-reactive withthe gold in the substrate and thereafter quickly deposited upon thesubstrate. Once the cell is filled, the reference electrode and thecounter .[.electrodes.]. .Iadd.electrode .Iaddend.are connected and astable layer of adsorbate is formed on the gold electrode where it canbe readily scanned with the microscope probe.

Accordingly, the principle object of the present invention is to providenew and improved methodology for preparing molecular adsorbates forscanning probe microscopy.

Another object of the present invention is to provide methodologyespecially adapted to potentiostatically prepared negatively chargedmolecular adsorbates for scanning probe microscopy.

These and still further objects as shall hereinafter appear are readilyfulfilled by the present invention in a remarkably unexpected manner aswill be readily discerned from the following detailed description of anexemplary embodiment thereof especially when read in conjunction withthe accompanying drawing .[.in which like parts bear like numeralsthroughout the several views.]..

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings:

FIG. 1.[...]. is a schematic drawing of a simple electrochemical cell.Iadd.according to the present invention.Iaddend.; and

FIG. 2 is a cyclic voltammogram of a solution used for deposition of DNAtaken in situ using a phosphate buffer solution adjusted to pH=6 withNaOH and containing .[.5 micrograms 5/ml.]. .Iadd.5 micrograms permilliliter .Iaddend.of DNA.

DESCRIPTION OF PREFERRED EMBODIMENT

A small electrochemistry cell is mounted on an STM or AFM as describedin my prior U.S. Pat. No. 4,868,396. A sketch of the current simplifiedcell is shown in FIG. 1. The substrate is Au (111), the counterelectrode a platinum wire and the reference electrode a silver wire. Ithas been discovered that silver wires produce results that are identicalto Ag/AgCl/KCl reference electrodes in these particular solutions, butare much easier to use and do not cause chlorine contamination of thesubstrate.

One practical arrangement of an electrochemistry cell is shown inFIG. 1. Referring to FIG. 1, the electrochemistry cell is designated bythe general reference 10 and comprises a gold-on-mica substrate11.[.,.]. .Iadd.and .Iaddend.a glass cell 12, having a polished bottomthat forms a seal against the gold substrate. A platinum (Pt) wirecounter electrode 13 and a silver (Ag) wire reference electrode 14extend into cell 12. Each of these wires are longer than needed and afresh cut surface is introduced into the cell for each experiment byadvancing the .[.electrode.]. .Iadd.electrodes .Iaddend.13, 14 in.[.its.]. .Iadd.their .Iaddend.respective electrode .[.holder.]..Iadd.holders .Iaddend.15, 15. A stainless steel plate 16 is glued tothe lower exterior surface of glass cell 12 to hold down the substrate11 and make electrical contact with the gold.

Cleanliness is critical. As will appear, an excess length of wire isused for each wire electrode. Before starting the next run, the usedportion of the wire is cut away and a portion of the fresh wire isadvanced into the cell. A fresh gold substrate is also used for eachrun.

In one practice of the present invention, a substrate is loaded onto theSPM. Clean reference and counter electrodes are placed into a cleanglass cell on the substrate as shown in FIG. 1. The polymer is dissolvedinto a buffer solution that does not react with the gold over theappropriate range of substrate potentials. One suitable buffer solutionfor use with Au (111) between -1.2 and .[.=.]. .Iadd.+ .Iaddend.1.3 V(vs. the Ag reference) is NaH₂ PO₄ .[...]. (10 mM adjusted to pH6 withNaOH). A cyclic voltammogram taken in situ is shown in FIG. 2. Forsparse coverage of the electrode, a solution that, at full adsorption,gives less .[.that.]. .Iadd.than .Iaddend.a monolayer coverage of themacromolecule is used. For example, in a 50 microliter cell (0.5 cm²electrode area) less than 5 micrograms of DNA per mL of solution arerequired.

The solution is placed onto the substrate as quickly as possible (tominimize contamination) and, once the cell is full, the reference andcounter electrodes are connected. Any positive potential (in case ofDNA) between the potentials at which reactions occur (from -0.2 V vs. Agto +0.6 V vs. Ag; the DNA bases oxidize at higher voltage) may beapplied to the substrate. There are some small reversible phosphate.[.absorptions.]. .Iadd.adsorptions .Iaddend.at lower potentials, but inthe double-layer region macromolecules can be seen in stablearrangements all the way up to about 1 volt. The voltage employed in anygiven reading is correlated to the reference electrode. The voltagerequired for deposition is dependant upon the salt solution used. Thevalues reported herein are for the phosphate buffer solution.

If the solutions are free of contamination and, in the case of the STM,the tip is well insulated, a very stable layer of .[.absorbate.]..Iadd.adsorbate .Iaddend.is formed on the gold electrode. It may bescanned in situ repeatedly with no sign of sample movement ordegradation. Indeed this is the salient feature of this invention,namely that .[.the.]. an adsorbate, when under potentiostatic control,is remarkably stable. Furthermore, the adsorbate layer may be lifted.[.on and off.]. .Iadd.off and placed back on .Iaddend.the electrodesurface at will simply by cycling the substrate potential between apositive value and -0.2 V (vs. Ag). Of course, these conditionsrepresent a much diminished disruption of the solvated structure of thepolymers compared to methods where the adsorbate is chemically reactedonto the substrate.

The coverage of the substrate, even with the simple layout shown in FIG.1, is remarkably homogeneous. The whole problem of molecular microscopyis now reduced to scanning an area large enough to contain a fewmolecules (as calculated from the expected coverage, given the cellgeometry and sample concentration) and then applying a suitablepotential. Furthermore, reactions and various dynamic processes may bestudied simply by allowing them to proceed in the cell (using componentsand a potential that avoid irreversible reactions) and then applying.[.and.]. .Iadd.an .Iaddend.attractive charge to the substrate.

Finally it should be noted that the problem of contamination is greatlyreduced. Only those molecules that satisfy conditions for physicaladsorption appear in the image. In contrast a vacuum or ambient imagewould show all contaminants.

This method can also be used to hold positively charged moleculesproviding the reaction current due to dissolved oxygen is eliminated.This may be done .Iadd.by .Iaddend.using degassed solutions and byoperating in an inert gas .[.enviroment.]. .Iadd.environment.Iaddend..

Experiments demonstrate that this method yields excellent highresolution images of macromolecular .[.absorbates.]. .Iadd.adsorbates.Iaddend.in both the STM and the AFM. The electrodes and buffers hereindisclosed are intended as representative preferred materials and not byway of limitation thereon.

From the foregoing, it is readily apparent that a useful embodiment ofthe present invention has been herein described and illustrated whichfulfills all of the .[.aforestated.]. .Iadd.aforementioned.Iaddend.objectives in a remarkably unexpected fashion. It is of courseunderstood that such modifications, alterations and adaptations as mayreadily occur to the artisan confronted with this disclosure areintended within the spirit of this disclosure which is limited only bythe scope of the claims appended hereto.

Accordingly, what is claimed is:
 1. A method of preparing molecularadsorbates for scanning probe microscopy comprising: loading a substrateinto a cell; placing the cell on a scanning probe microscope; placing aclean reference electrode in said cell; placing a clean counterelectrode in said cell in spaced relationship to said referenceelectrode; dissolving a polymer containing negatively charged moleculesinto a buffer solution that is inert relative to said substrate; fillingsaid cell with said polymer .[.contained.]. .Iadd.containing.Iaddend.buffer solution; activating said reference electrode and saidcounter electrode and applying a potential to said substrate to depositand secure said polymer onto said substrate for examination by saidmicroscope.
 2. A method according to claim 1 in which said substrate isgold (111).
 3. A method according to claim 1 in which said referenceelectrode is silver wire.
 4. A method according to claim 1 in which saidcounter electrode is platinum wire.
 5. A method according to claim 1 inwhich said buffer solution is NaH₂ PO₄ at a pH of
 6. 6. A methodaccording to claim 3 in which said electrodes are activated to a voltageof from about -1.2 up to about .Iadd.+ .Iaddend.1.3.
 7. A methodaccording to claim 5 in which said electrodes are activated to a voltageof from about -1.2 up to about .Iadd.+ .Iaddend.1.3.
 8. A methodaccording to claim 2 in which said reference electrode is silver wire.9. A method according to claim 8 in which said counter electrode isplatinum wire.
 10. A method according to claim 9 in which said buffersolution is NaH₂ PO₄ at a pH of
 6. 11. A method according to claim 10 inwhich said electrodes are activated to a voltage of from about -1.2 upto about .Iadd.+ .Iaddend.1.3. .Iadd.
 12. A method of preparingmolecular adsorbates for scanning probe microscopy comprising:loading asubstrate into a cell; placing the cell on a scanning probe microscope;placing a clean reference electrode in said cell; placing a cleancounter electrode in said cell in spaced relationship to said referenceelectrode; dissolving a polymer containing charged molecules into abuffer solution that is inert relative to said substrate; filling saidcell with said polymer containing buffer solution; activating saidreference electrode and said counter electrode; and applying a potentialbetween said substrate and said counter electrode to deposit and securesaid polymer onto said substrate for examination by saidmicroscope..Iaddend..Iadd.13. A method according to claim 12 in whichsaid substrate comprises gold..Iaddend..Iadd.14. A method according toclaim 12 in which said reference electrode is silverwire..Iaddend..Iadd.15. A method according to claim 12 in which saidcounter electrode is platinum wire..Iaddend..Iadd.16. A method accordingto claim 12 in which said buffer solution is NaH₂ PO₄ at a pH of 6..Iaddend..Iadd.17. A method according to claim 13 in which saidsubstrate is activated to a voltage in a range of about -1.2 volts up toabout +1.3 volts with respect to said reference electrode by applicationof a suitable voltage to said counter electrode..Iaddend..Iadd.18. Amethod according to claim 14 in which said substrate is activated to avoltage in a range of about -1.2 volts up to about +1.3 volts withrespect to said reference electrode by application of a suitable voltageto said counter electrode..Iaddend..Iadd.19. A method according to claim15 in which said substrate is activated to a voltage in a range of about-1.2 volts UP to about +1.3 volts with respect to said referenceelectrode by application of a suitable voltage to said counterelectrode..Iaddend..Iadd.20. A method according to claim 16 in whichsaid substrate is activated to a voltage in a range of about -1.2 voltsup to about +1.3 volts with respect to said reference electrode byapplication of a suitable voltage to said counterelectrode..Iaddend..Iadd.21. A method according to claim 13 in whichsaid reference electrode is silver wire..Iaddend..Iadd.22. A methodaccording to claim 15 in which said reference electrode is silverwire..Iaddend..Iadd.23. A method according to claim 13 in which saidcounter electrode is platinum wire..Iaddend..Iadd.24. A method accordingto claim 16 in which said counter electrode is platinumwire..Iaddend..Iadd.25. A method according to claim 23 in which saidbuffer solution is NaH₂ PO₄ at a pH of
 6. .Iadd.26. A method accordingto claim 25 in which said substrate is activated to a voltage in a rangeof about -1.2 volts up to about +1.3 volts with respect to saidreference electrode by application of a suitable voltage to said counterelectrode..Iaddend..Iadd.27. An electrochemical fluid cell and substrateassembly for use in studying molecular adsorbates with a scanning probemicroscope, said cell comprising:an electrically conductive substrate;an electrically insulating cell wall having an inner boundary defining afluid container open at its top, said fluid container having a bottomboundary defined by said substrate; said substrate having a portionextending under and beyond said inner boundary; a reference electrodeextending from beyond said fluid container into said fluid containerthrough said top; a counter electrode extending from beyond said fluidcontainer into said fluid container through said top and maintained inspaced relationship to said reference electrode; a voltage potentialcapable of being applied to said substrate by connecting said voltagepotential to said electrically conductive substrate at said portionextending under and beyond said inner boundary..Iaddend..Iadd.28. Anelectrochemical fluid cell and substrate assembly according to claim 27wherein said substrate comprises gold..Iaddend..Iadd.29. Anelectrochemical fluid cell and substrate assembly according to claim 27wherein said reference electrode is silver wire..Iaddend..Iadd.30. Anelectrochemical fluid cell and substrate assembly according to claim 27wherein said counter electrode is platinum wire..Iaddend..Iadd.31. Anelectrochemical fluid cell and substrate assembly according to claim 28wherein said reference electrode is silver wire..Iaddend..Iadd.32. Anelectrochemical fluid cell and substrate assembly according to claim 28wherein said counter electrode is platinum wire..Iaddend..Iadd.33. Anelectrochemical fluid cell and substrate assembly according to claim 28wherein said reference electrode is silver wire and said counterelectrode is platinum wire..Iaddend..Iadd.34. An electrochemical fluidcell and substrate assembly for use in studying molecular adsorbateswith a scanning probe microscope, said cell comprising:an electricallyconductive substrate; an electrically insulating cell wall having aninner boundary defining a fluid container open at its top, said fluidcontainer having a bottom boundary defined by said substrate; saidsubstrate in electrical contact with a conductor having a portionextending under and beyond said inner boundary; a reference electrodeextending from beyond said fluid container into said fluid containerthrough said top; a counter electrode extending from beyond said fluidcontainer into said fluid container through said top and maintained inspaced relationship to said reference electrode; a voltage potentialcapable of being applied to said substrate by connecting said voltagepotential to said electrically conductive substrate at said portion ofsaid conductor extending under and beyond said innerboundary..Iaddend..Iadd.35. An electrochemical fluid cell and substrateassembly according to claim 34 wherein said substrate comprisesgold..Iaddend..Iadd.36. An electrochemical fluid cell and substrateassembly according to claim 34 wherein said reference electrode issilver wire..Iaddend..Iadd.37. An electrochemical fluid cell andsubstrate assembly according to claim 34 wherein said counter electrodeis platinum wire..Iaddend..Iadd.38. An electrochemical fluid cell andsubstrate assembly according to claim 35 wherein said referenceelectrode is silver wire..Iaddend..Iadd.39. An electrochemical fluidcell and substrate assembly according to claim 35 wherein said counterelectrode is platinum wire..Iaddend..Iadd.40. An electrochemical fluidcell and substrate assembly according to claim 35 wherein said referenceelectrode is silver wire and said counter electrode is platinum wire..Iaddend.